The present invention relates to nucleotide-sugar-synthesizing enzymes, i.e. enzymes with nucleotidyltransferase or pyrophosphorylase activity, from nonparasitic protists, to a process for the preparation thereof and to the use thereof for preparing nucleotide-sugars.
Oligo- and polysaccharides play an important part in nature. They are of essential importance as components of glycoproteins and glycolipids in cellular communication processes and as binding sites for, for example, antibodies, lectins and hormones. They also crucially determine the physical properties of proteins such as solubility, stability and 3D structure. They furthermore act as receptors for pathological states mediated by viruses, bacteria and protists.
The recognition of the versatile functions of the carbohydrate residues in glycoconjugates is also associated with an increasing interest in synthesizing such compounds. However, because of their great complexity and the specific requirements for chemoselectivity, regioselectivity and stereoselectivity, the synthesis even of lower oligosaccharides by chemical means is extremely difficult. As the complexity of the saccharides increases there is also an increase in the difficulty of reproducing them by chemical synthesis. For this reason it is obvious to copy the biological synthetic routes and to have recourse to enzymes, which usually catalyze substrate-, regio- and stereospecific reactions, for synthesizing complex saccharides.
The oligosaccharide structures of glycoconjugates are synthesized in vivo by Leloir glycosyltransferases which require activated sugars (nucleotide-sugars) as donor substrates for the regio- and stereoselective transfer. The nucleotide-sugars are produced by nucleotidyltransferases or pyrophosphorylases (for example EC 2.7.7.9 to 2.7.7.13, 2.7.7.23, 2.7.7.29, 2.7.7.30) using nucleoside triphosphates. In this, transfer of the nucleotidyl group of a nucleoside triphosphate (NTP) to a sugar phosphate (sugar(-1-P)) is catalyzed with liberation of pyrophosphate (PPi).
The use of oligosaccharide structures as potential therapeutic agents (inhibitors, antiadhesive agents, antiinflammatory agents, immunogenic stimulators etc.) or diagnostic aids (determinants, lectin ligands) requires unrestricted synthetic access thereto.
There are in principle two possible approaches to the synthesis of oligosaccharide structures; a chemical and an enzymatic synthesis. Chemical synthesis is associated with many disadvantages because complex strategies using protective group techniques are, as a rule, time-consuming and result in only low yields. This applies equally to the chemical preparation of nucleotide-sugars. However, the costs are very high because of the elaborate synthesis and the frequently poor yields. Thus, 10 mg of chemically synthesized GDP-β-L-fucose cost about 1000 DM.
A number of glycosyl transferases are now available for enzymatic synthesis of oligosaccharide structures. More than one hundred glycosyl transferases have now been isolated from eukaryotes (Glycobiology, 1995, 5, 463). A crucial precondition for industrial synthesis is enzymatic access to the costly nucleotide-sugars. There is at present a great demand and great scientific interest therein. Thus, for example, enzymatic synthesis of GDP-β-L-fucose has not been possible on the industrial scale in the absence of a suitable enzyme system.
The object therefore is to find novel “microbial” sources which can be fermented on a large scale for obtaining nucleotide-sugar-synthesizing enzymes. Surprisingly, it has been possible in this connection to identify a group of organisms which has hitherto been completely ignored—the free-living, nonparasitic protists—as novel and promising producers of the enzymes which are sought.
Hitherto, only a few pyrophosphorylases were known from parasitic protists, either pathogens or facultative pathogens, such as, for example UDP-N-acetylglucosamine pyrophosphorylase from Giardia (Mol Biochem. Parasitol., 1992, 56, 301), UTP-hexose-1-phosphate uridyltransferase (E.C. 2.7.7.10) and UDP-glucose pyrophosphorylase from Entamoeba histolytica (Mol. Biochem. Parasitol., 1983, 7, 173 and Exp. Parasitol., 1977, 43, 115), UDP-glucose pyrophosphorylase from Hartmanella culbertsoni (J. Protozool., 1971, 18, 115) and UDP-glucose pyrophosphorylase from Acanthamoeba castellanii (J. Biol. Chem., 1974, 249, 7832).
Apart from the UDP-glucose pyrophosphoryltase from Tetrahymena pyriformis (Arch. Biochem. Biophys., 1968, 127, 72) and from Dictyostelium (J. Paulovic et al., Dev. Genet. 9, 371–382 (1988); J. A. Ragheb et al., Nucleic Acid Res. 15, 3891–3896 (1987)), astonishingly no nucleotidyl transferases have hitherto been described in free-living, nonparasitic protists.